Tuberculosis (TB) is the leading cause of death worldwide from an infectious agent (Flint et al., 2004), with the WHO estimating that one third of the global population are infected with TB. In a global report from the WHO (2009), it was estimated that there were 9.27 million cases of TB in 2007, with 2 million associated deaths. TB in mammals is caused by members of the Mycobacterium tuberculosis Complex (MTC). The eight closely related species in the complex have a wide range of natural hosts including humans hosts (M. tuberculosis M. africanum M. canetti), bovine hosts (M. bovis), caprine hosts (M. caprae), rodent hosts (M. microti) and pinniped hosts (M. pinnipedii) along with the attenuated M. bovis strain BCG (Bacillus Calmette-Guérin), the commonly used vaccine strain. While there are a number of natural hosts, each member of the MTC has been implicated in human infection (Brosch et al., 2002; Kiers et al., 2008a).
Traditionally, diagnosis of TB relies on culture techniques and a battery of biochemical tests which are time consuming, labour intensive and often yield insensitive results (Huard et al., 2003). Nucleic Acid Diagnostics (NAD), in particular real-time PCR, offer a rapid, reliable and highly sensitive alternative diagnostic tool for many infectious agents (Malhotra-Kumar et al., 2008; Yang & Rothman, 2004). Advances in real-time PCR such as the availability of multiple fluorophores, along with the development of non-fluorescent quenchers has facilitated multiplexing, allowing for the simultaneous detection and discrimination of multiple targets, along with internal controls, in one reaction (Arya et al., 2005).
While significant advances have been made in the diagnosis of TB using NAD (Huard et al., 2006), the differentiation of members of the MTC to the species level is not routinely performed. Conventional PCR and real-time PCR assays for the rapid diagnosis of the MTC have been described (Huard et al., 2003; Parsons et al., 2002). Also, commercially available real-time PCR kits for the diagnosis of TB are available, such as AMPLIFIED MTD (Gen-Probe, San Diego, Calif.), Xpert MTB/RIF (Cepheid, Sunnyvale, Calif.) and AMPLICOR MTB (Roche, Branchburg, N.J.). These kits identify the MTC, but not individual species.
The high degree of nucleotide sequence homology between members of the complex makes discrimination of species challenging, which may explain why it is not routinely carried out (Pinsky & Banaei, 2008). Comparative genomics revealed that M. tuberculosis and M. bovis genomes are 99.95% similar (Garnier et al., 2003), with whole genome DNA microarrays identifying 16 regions of difference (RD 1-16). (Behr et al., 1999). These RDs represent regions of the genome deleted in M. bovis BCG which are present in M. tuberculosis and have been used for the differentiation of members of the MTC. One RD commonly targeted for the specific detection of M. tuberculosis is RD9 (Pinsky & Banaei, 2008), however this RD is also present in M. canettii (Brosch et al., 2000). There is currently no real-time PCR test which can diagnose TB, whilst identifying the exact causative agent of infection.
Differentiation of the MTC allows health care professionals to determine the most appropriate course of treatment for infected patients and also provides valuable epidemiological information with relation to prevalence, transmission and geographical distribution of the neglected members of the MTC including members associated with zoonotic TB infection in humans. There is currently one molecular based kit commercially available for differentiation of the MTC, the GenoType MTBC (Hain Lifesciences GmbH, Nehren, Germany). However this kit is unable to differentiate between M. tuberculosis and M. canettii or between the two clades of M. africanum, and the target used in this kit for the detection of M. africanum also crossreacts with M. pinnipedii. 
M. tuberculosis is the most important human pathogen in the MTC and is thought to be responsible for 95% of human cases of TB, yet rarely causes disease in other mammals (Brosch et al., 2000; Das et al., 2007). While drug resistant strains of M. tuberculosis are emerging, it is considered sensitive to anti-tuberculosis drugs such as Pyrazinamide (PZA), a first line antibiotic that reduces patient treatment time from 9 months to 6 months (Niemann et al., 2000; Somoskovi et al., 2006).
M. canettii is thought to be the most phylogenetically distant member of the MTC and is considered the species from which other members of the complex may have evolved (Brosch et al., 2002). M. canettii is phenotypically characterised by its smooth glossy white colonies, however a small number of these colonies have been shown to revert to rough colony variants when individual colonies are replated (van Soolingen et al., 1997). Smooth colonies are uncharacteristic of the MTC and are due to the presence of large amounts of lipooligosaccharides in the M. canettii cell wall (Pfyffer et al., 1998). Like M. tuberculosis, M. canettii contains all the RDs with the exception of RD12 canettii (RD12can) which has been targeted for the specific detection of M. canettii in a complicated conventional PCR approach (Huard et al., 2003). The method provided by Huard et al. requires time-consuming multiple reactions and produces results that require detailed interpretation. To achieve the limited distinction that the methods of Huard et al. and other methods of the prior art offer, detailed analysis of gels must be undertaken. This requires that polyacrylamide gels, for example, are prepared and run and then analysed by eye.
While infection with M. canettii is thought to be rare, there is a lack of rapid diagnostic tests available to differentiate between M. tuberculosis and M. canettii. Also recent reports have suggested that the true cases of TB caused by M. canettii may in fact be underrepresented (Goh et al., 2001; Somoskovi et al., 2009). While differentiation between M. tuberculosis and M. canettii is useful from an epidemiological point of view, it is also important for indicating the therapeutic approach to treatment as M. tuberculosis is sensitive to PZA, whereas M. canettii is resistant (Somoskovi et al., 2009).
The major ethologic agents of zoonotic TB in humans are the phylogenetically related species M. bovis and M. caprae. These species occur worldwide and there are indications which suggest the true prevalence of zoonotic human TB infection may be underrepresented (Ojo et al., 2008; Cicero et al., 2009; Allix-Beguec et al. 2010). In developed countries it has been suggested that the burden of bovine TB in humans ranges from 0.5 to 7.2% of TB cases, while in developing countries, where very little data are available, this figure may be up to 15% (de la Rua-Domenech, 2006; Kubica et al., 2003). Recent reports have identified TB in humans caused by M. bovis in countries officially free from bovine TB and suggest that the true prevalence of zoonotic TB may be underestimated clinically (Cicero et al., 2009; Allix-Beguec et al., 2010). Moreover, zoonotic TB remains a significant threat to human health in developing countries where its prevalence is currently unknown, as speciation of the MTC is not routinely performed (de la Rua-Domenech, 2006). M. bovis and M. bovis BCG are intrinsically resistant to pyrazinamide (PZA), and this important first line drug for treating disease caused by M. tuberculosis and M. caprae infection should not be used for treating M. bovis, or M. bovis BCG infection. It is therefore important to distinguish between these members of the MTC in order to provide a useful treatment regimen.
This invention provides a multiplex in vitro nucleic acid amplification assay using novel nucleic acid targets which can diagnose TB from clinical isolates by detecting the MTC while simultaneously differentiating between the different species that are members of the MTC.